Peptides and uses thereof

ABSTRACT

The invention provides a series of peptide signals which, when linked to a polypeptide of interest (POI), ensure that said polypeptide is secreted in high yields by a host cell such as  Mycoplasma pneumoniae . The invention also provides fusion proteins tagged with said peptide signals, the nucleic acid sequences coding for them, host cells comprising said tagged fusion proteins and a variety of uses of the fusion proteins and the host cells.

The present invention provides peptides which, when fused to apolypeptide of interest, drive the efficient secretion of thepolypeptide of interest out of bacterial cells. The invention hasmultiple potential applications, in particular in the area ofnext-generation bacterial-based therapies.

BACKGROUND ART

Secretory protein expression is the expression of a protein in a hostcell, where the protein is exported trough the cell membrane for itsrelease into the extracellular medium or its displayed on the cellsurface, anchored to the cell membrane. Secretory protein expression ismediated by a signal peptide at the N-terminus of the protein, whichdirects the extracellular export of the polypeptide.

Usually, recombinant proteins are intracellularly produced inprokaryotic hosts. When the protein is recovered in such a procedure,the cells have to be lysed which leads to contamination of therecombinant protein with cellular content. The protein then has to berecovered from whole cell extracts in multi-step purificationprocedures, which is time consuming and results in poor yields. Alsosecreted proteins could be used for bacterial therapy where they targeteukaryotic receptors on the target cells.

Secretion of recombinant proteins into the medium is a better strategybecause purification of proteins from spent medium is easier and morecompatible with continuous culturing.

Secretory protein expression has other uses. Examples of use for thistype of protein expression include live-vaccine development, epitopemapping, biosorbent and biosensor development and the high throughputscreening of protein and peptide libraries for drug discovery.

In secretion, recombinant proteins face the challenge of translocationacross the complex cell envelope that consists of two lipid membranes(the inner and outer membrane) with a gel-like compartment, theperiplasm, in between. This has been shown to be very difficult and themethods previously used have had low efficacy.

On the other hand, synthetic biology is based on engineering livingorganisms in order to exploit them in a myriad of applications rangingfrom biosensors and the production of biofuels to therapeutictreatments. Microorganisms have now long been used by the industry inthe production of many therapeutic proteins. However, the fine tuning ofa whole microorganism in terms of its genome by a synthetic biologyapproach opens up a way of exploiting the organism itself as a therapyor a therapeutic vehicle. One particularly exciting application is thepossibility of creating bacterial factories that can live in diseasedtissues and produce, display or secrete therapeutic proteins in situ.

In order to manipulate a host cell such as a bacterium and to turn itinto a therapeutic vehicle, one must first have a very deepunderstanding about its genome, its proteome and all its metabolicprocesses. Currently a main bottleneck is the inability to predict withaccuracy the behaviour of engineered bacteria. In order to minimizepotential undesired effects in therapeutic applications, and with a goalof simplifying a very complex living system, some of the simplestbacteria are being studied as synthetic biology platforms.

Host bacteria can be engineered so that they synthesize a polypeptide ofinterest (POI) with therapeutic applications. With the goal ofincreasing the surface display to the exterior of the cell and/or theirsecretion to the outer medium, a series of strategies can beimplemented. One of them is the coupling (fusion) in frame of thepolypeptide of interest with a second peptide that promotes a variety ofsecretion processes within the host cell. Some peptides can drive POI'ssurface display in the extracellular section of the cell membrane. Inthis case, the POI can be used for eliciting an immune response via thedisplay of a foreign protein to an immune system's machinery, andtherefore the engineered bacteria carrying the fusion protein can beused as a vaccine. Some other peptides can drive the production andsecretion of the fusion protein (POI-secretion enhancer) to the outermedium. In this latter case, the engineered bacteria can be used as atissue delivery chassis of therapeutic proteins.

A range of references exist in the prior art that deal with peptidesuseful in surface displaying and secreting intended polypeptides.However, in spite of what is known in the field, there are many issuesthat still need to be addressed. The successful secretion of apolypeptide of interest by a host cell is still hindered by many factorssuch as: (a) the coupling of the secretion enhancer to the POI can alterthe folding and final structure of either or both partners, with asubsequent change in their biological functions; and (b) the surfacedisplay and/or secretion yields are suboptimal in terms of realapplicability for many bacterial host cells such as Mycoplasma.

Therefore, it is desirable to provide for other peptide secretionenhancers.

SUMMARY OF THE INVENTION

Inventors have found a series of secretion signal peptides that, whenfused to a POI, drive its efficient secretion in a bacterial host, suchas Mycoplasma pneumoniae, and do not interfere with their biologicalactivity, as seen in the experimental data below. Surprisingly,inventors have found that these peptides are effective in secreting POIswith a very wide range of protein folds. This reveals that the signalpeptides of the invention have a wide applicability spectrum in terms ofsecretion of POIs.

Due to this efficient secretion profile, the POI purification from themedium is easier and more compatible with continuous culturing.

Thus, a first aspect of the present invention is a peptide comprising asequence which has at least 90% of homology with one of the sequences ofthe group consisting of:

Mpn 142 with sequence:  SEQ ID NO: 1 MKSKLKLKRYLLFLPLLPLGTLSLANTY;Mpn 645 with sequence:  SEQ ID NO: 2 MKLKLKFLLISLLGSSLLLSACSSAATQ;Mpn 400 with sequence: SEQ ID NO: 3MKLNFKIKDKKTLKRLKKGGFWALGLFGAAINAFSAVL; Mpn 200 with sequence: SEQ ID NO: 4 MKFKYGAIVFSGLLGVSAILAACGT; Mpn 213 with sequence: SEQ ID NO: 5 MKLSAIISLSVAGTVGTTAVVVPTTITLVNK; Mpn 489 with sequence: SEQ ID NO: 6 MGYKLKRWPLVAFTFTGIGLGVVLAACSALN;

The peptides of the invention can be either fused in frame directly tothe POI, or can be fused with the POI via a linker. The linker can havea series of properties. For instance, it can be a cleavage signal sitewhich is the substrate of proteases, so that the cleavage of the POI andthe peptides of the invention can be controlled under certaincircumstances (presence or absence of the protease capable of processingthe cleavage site) if it is needed.

As it is shown below, the peptide of the invention can be fused to aparticular POI in order to efficiently secrete it in a particularbacterium. As it is illustrated below (see examples section and FIGS. 1to 3), when a POI is endowed with a certain biological activity, theresulting fusion protein (POI-peptide of the invention) is also active.This is indicative that the peptide of the first aspect of the inventiondoes not negatively affect the activity of the POI.

Therefore, in a second aspect the present invention provides a fusionprotein comprising a polypeptide of interest (POI) and at least onepeptide as defined in the first aspect of the invention, wherein thepolypeptide of interest is heterologous to the at least one peptide.

A third aspect of the present invention is a nucleotide sequence codingfor the peptide of the first aspect of the invention or the fusionprotein of the second aspect of the invention.

Once the nucleotide sequence coding either the peptide of the firstaspect or the fusion protein of the second aspect is generated, it isintegrated in a suitable vector, which then is integrated in a host cellin order to express and secrete the desired fusion protein. Thus, afourth aspect of the invention is a vector comprising the nucleotidesequence of the third aspect of the invention.

A fifth aspect of the invention is a host cell comprising the vector ofthe fourth aspect of the invention.

In addition, as it is illustrated in by the experimental data and theexamples, once generated the fusion protein, the bacterium is able ofefficiently secrete the active POI.

The modified host cell of the present invention, which has beenengineered for expressing the fusion protein POI+signal peptide of theinvention, can be effectively used as a bacterial factory, which canlive in diseased tissues and produce and secrete the active POI in situ.For this therapeutic in situ application, the host cell has to beselected from among those recognized as safe and non-toxic for humansand non-human animals. Alternatively, the cell can be engineered inorder to make it safe and non-toxic. This therapeutic application ispossible in cases where POI is a therapeutic peptide and the bacterialfactory is used as a factory of the therapeutic protein.

If the host cell is engineered to be safe and non-toxic, it can itselfbe administered in the treatment of disease. The host cell can beengineered to be able to colonize and in if desired reproduce safely ina certain tissue, propagating in it and secreting the therapeutic fusionprotein.

In a sixth aspect the present invention provides a pharmaceutical orveterinary composition comprising the fusion protein as defined in thesecond aspect of the invention or the host cell as defined in the fifthaspect of the invention, the host cell being safe and non-toxic forhumans or non-human animals, together with pharmaceutically acceptableexcipients or carriers.

In a seventh aspect the present invention provides the use of thepeptide of the first aspect of the invention as secretion peptide of apolypeptide of interest (POI).

In an eighth aspect the present invention provides a fusion protein asdefined in the second aspect of the invention or a host cell as definedin the fifth aspect of the invention, the host cell being safe andnon-toxic for human or non-human animals, for use in therapy.

In an ninth aspect the invention provides a method for secretory proteinexpression of a fusion protein as defined in the second aspect of theinvention, comprising the steps of: (a) providing a host cell accordingto the fifth aspect of the invention; and (b) inducing expression of thefusion protein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Represents the secretion of p53 by different modified M.pneumoniae (a=Mpn-142-p53, b=Mpn-645-p53, c=Mpn-400-p53, d=Mpn-459-p53,e=Mpn-332-p53, f=Mpn-200-p53, g=Mpn-506-p53, h=Mpn-489-p53) into McCoyMedia. X-axis=time after inoculation (t) expressed in hours;Y=concentration of p53 (pg/mL). The amounts were determined using aRoche p53 pan ELISA sandwich assay.

FIG. 2 Represents a bar-representation showing the alginate lyaseactivity assay in full medium supernatant from cultures secretingalginate lyase (M129 strain). All cultures were normalized to the sameinoculation count at day 0 (bar “a”) and over the next 5 days samples ofthe media were taken (bar “b”=sample taken at day 1, bar “c”=sampletaken at day 2, and bar “d”=sample taken at day 5). The assay is basedon the degradation of polymeric alginate to shorter sugar fragments.

FIG. 3_(A) represents the amount of secreted A1AT (ng/mL) by differentstrains of M. pneumoniae (specified in X-axis) cultivated in minimalmedium (grey bar) or full Hayflick Media (black bar); (B) shows aneutrophil elastase activity assay (Y-axis, in rfu) using concentratedminimal media supernatant culture from MP WT and MP142 strains: A=MediaControl, B=Concentrated M. pneumoniae supernatants, C=Reaction bufferonly, (1)=NE (8 nM), (2)=NE+A1AT, (3)=MP-WT, (4)=MP142; (c) shows aneutrophil elastase activity absorbance (N.E.A.A.) using purified A1ATfrom MP142 strain and purified A1AT from E. Coli (Acrys). Stripped barsand thick bars represent different biological replicas.

DETAILED DESCRIPTION OF THE INVENTION

As mentioned above, a first aspect of the invention is a peptidecomprising a sequence having at least 90% of homology with one of thesequences of the group consisting of: SEQ ID NO: 1; SEQ ID NO: 2; SEQ IDNO: 3; SEQ ID SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6.

In the context of the invention, the term “signal peptide”, alsoreferred to as “secretion enhancer”, is a short peptide (from 5 to 30amino acids long) which is fused to the POI and allows the secretion ofa particular POI to the extracellular medium.

In one embodiment of the first aspect of the invention the peptidecomprises a sequence having at least 90% of identity with one of thesequences of the group consisting of: SEQ ID NO: 1; SEQ ID NO: 2; SEQ IDNO: 3; SEQ ID

NO: 4; SEQ ID NO: 5; SEQ ID NO: 6.

The “percentage of homology”_between two amino acid sequences or twonucleotide sequences is to be understood as the percentage of identicalsequence positions or replaced with other amino acids with side chainsof similar features (i.e. polar, non-polar, with amino groups, with —SHgroups), or other nucleotides, according to the broadly acceptedclassifications known by an expert in the field. The “percentage ofidentity” between two amino acid sequences or two nucleotide sequencesis to be understood as the percentage of the sequence positions withidentical amino acids or nucleotides. The percentage of homology and ofidentity between sequences may be calculated by means of “sequencealignment”. The sequence alignment may be local or global. In the senseof the present invention the percentage of homology and of identity willbe calculated, preferably, over a global alignment, among the entiresequence or an entire active fragment of the sequence. Global alignmentsare more useful when the sequences are similar and have approximatelythe same size (length). There are several algorithms available in thestate of the art for performing these global alignments. There are alsobioinformatics tools using such algorithms to obtain the percentage ofidentity and homology between sequences. As an example, global alignmentbetween sequences may be performed by means of the well-known GGSEARCHor GLSEARCH software.

In another embodiment of the first aspect of the invention the peptideis one consisting in a sequence having at least 90% of homology with oneof the sequences of the group consisting of: SEQ ID NO: 1; SEQ ID NO: 2;SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6.

In another embodiment of the first aspect of the invention the peptideis one consisting in a sequence having at least 90% of identity with oneof the sequences of the group consisting of: SEQ ID NO: 1; SEQ ID NO: 2;SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6.

In still another embodiment the % of homology is of at least 90, 91, 92,93, 94, 95, 96, 97, 98, 99 or 100.

In still another embodiment the % of identity is of at least 90, 91, 92,93, 94, 95, 96, 97, 98, 99 or 100.

In one embodiment the peptide consists of a sequence which is one of thesequences of the group consisting of: SEQ ID NO: 1; SEQ ID NO: 2; SEQ IDNO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6.

In another embodiment of the first aspect of the invention, the peptidecomprises or consists of the sequence SEQ ID NO: 1, SEQ ID NO: 2; SEQ IDNO: 3. Advantageously, these peptides are the ones giving the mostefficient results in terms of secretion of the active POI, as shown inthe experimental data found below.

In a second aspect, the present invention provides a fusion proteincomprising at least one POI together with a peptide as defined in thefirst aspect of the invention.

The term “fusion protein” as used herein is the result of in framecoupling of a polypeptide of interest (POI) through either its N- orC-terminal end to a peptide of the first aspect of the invention.

The term “polypeptide of interest” or simply POI, as used herein, is apolypeptide that the user of the invention wants a host cell to secretein soluble form into the medium. Typically, the POI is a protein thatthe user wants to have secreted, whereas the signal peptide of thefusion protein assists in the secretion process. Typically, the POI isalso heterologous to the signal peptide to which it is fused, whichmeans that the POI does not originate from the same species as thesignal peptide.

In one embodiment of the second aspect of the invention, the fusionprotein comprises from 1 to 12 POIs. In another embodiment of the secondaspect of the invention, the fusion protein comprises from 1 to 5 POIs.In still another embodiment of the second aspect of the invention, thefusion protein comprises 1 POI.

In one embodiment of the second aspect of the invention, POI has atherapeutic effect such that, when administered in a therapeuticallyeffective amount to a patient suffering from a disease, it has abeneficial effect on the health and well-being of the patient, eithercuring the disease, slowing the progress of the disease; causing thedisease to regress; alleviating one or more symptoms of the disease;preventing the occurrence of a disease or disorder; retarding therecurrence of the disease or disorder; or protecting against the onsetof the disease.

Illustrative non-limitative examples of therapeutic POIs areantiproliferative agents, antiinflammatory agents, antineoplasticagents, antimitotic agents, antiplatelet agents, anticoagulant agents,antifebrin agents, antithrombin agents, cytostatic agents, antibiotics,angiogenic agents, hormones, and antigens. Alternatively, the POI can bea non-therapeutic polypeptide, such as an enzyme with a particularindustrial interest (such as in fermentation processes, catalysis,etc.).

In another embodiment the POI is p53, Alginate Lyase Al-III oral-antitrypsin.

In another embodiment of the second aspect of the invention, theN-terminal end of the POI is bound to the peptide of the first aspect ofthe invention through a peptide linker. This linker (or “spacer”) makesit more likely that POI and signal peptide fold independently and behaveas expected; i.e., incorporating a linker it is guaranteed that POIretains its function. In addition, this linker can be a cleavage signalsite which is the substrate of proteases, so that the cleavage of thePOI and the peptides of the invention can be controlled under certaincircumstances (presence or absence of the protease capable of processingthe cleavage site) if it is needed.

The terms “peptide linker” and “linker” are used interchangeably, andhas to be understood as any amino acid sequence comprising from 1 to 100amino acids, such sequence not negatively affecting neither POI'sactivity nor signal peptide function as displaying or secreting tool.

In another embodiment of the second aspect of the invention, the peptidecomprises a sequence having at least 90% of homology with one of thesequences of the group consisting of: SEQ ID NO: 1; SEQ ID NO: 2; SEQ IDNO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6. In another embodimentof the second aspect of the invention, the peptide comprises a sequencehaving at least 90% of identity with one of the sequences of the groupconsisting of: SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4;SEQ ID NO: 5; SEQ ID NO: 6. In another embodiment of the second aspectof the invention the peptide is one consisting in a sequence having atleast 90% of homology with one of the sequences of the group consistingof: SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO:5; SEQ ID NO: 6. In another embodiment of the second aspect of theinvention the peptide is one consisting in a sequence having at least90% of identity with one of the sequences of the group consisting of:SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5;SEQ ID NO: 6.

In another embodiment of the second aspect of the invention, the peptideconsists of a sequence selected from the group consisting of: SEQ ID NO:1; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6.In another embodiment of the second aspect of the invention, the peptideof the first aspect of the invention is selected from the groupconsisting of: sequence SEQ ID NO: 1, SEQ ID NO: 2; SEQ ID NO: 3; SEQ IDNO: 4; SEQ ID NO: 5 or SEQ ID NO: 6.

In a third aspect the present invention provides a nucleotide sequencecoding a peptide as defined in the first aspect of the invention or afusion protein as defined in the second aspect of the invention.

There are well-known techniques in the state of the art for obtainingthe nucleotide sequence from an amino acid sequence. When doing suchsequencing it is of particular relevance the cell wherein the expressionwill take place. As it is well-known, the genetic code is degenerate, sothere can be more than one nucleotide sequence coding for the same aminoacid sequence. The use of codons varies depending on the host cell interms of expression efficiency. The nucleotide sequence may in principlebe optimized for increased expression depending on the host cell. As itis explained below, the nucleotide sequence coding for peptide sequenceSEQ ID NO:1 has been optimized for expression in M. pneumoniae.

In one embodiment of the third aspect of the invention, the peptide ofthe first aspect of the invention is coded by a nucleotide sequencecomprising a sequence having at least 90% of homology with one of thesequences of the group consisting of: SEQ ID NO: 7 to SEQ ID NO: 11:

SEQ ID NO: 7 ATGAAGTCCAAGTTGAAACTCAAACGCTATTTACTCTTTCTCCCCTTGTTACCACTCGGTACCTTGAGTTTAGCTAACACTTAC SEQ ID NO: 8ATGAAATCGAAGCTAAAGTTAAAACGTTATTTACTGTTTTTACCACTTTTACCGCTAGGGACGTTGTCACTAGCCAACACCTAC SEQ ID NO: 9ATGAAACTGAAACTTAAATTTCTATTAATTTCTCTTTTAGGTTCTAGTTTGTTGTTAAGCGCTTGTTCTTCAGCAGCTACTCAA SEQ ID NO: 10ATGAAATTTAAGTATGGTGCCATTGTTTTCAGTGGTCTTTTAGGAGTCTCTGCCATTTTAGCTGCTTGTGGTACA SEQ ID NO: 11ATGAAGCTTAGTGCTATTATCTCCCTATCAGTCGCTGGTACTGTGGGAACAACTGCGGTGGTAGTACCTACAACTATAACGCTTGTAAATAAG

In another embodiment of the third aspect of the invention, the peptideof the first aspect of the invention is coded by a nucleotide sequencecomprising a sequence having at least 90% of identity with one of thesequences of the group consisting of: SEQ ID NO. 7 to SEQ ID NO: 11. Inanother embodiment of the third aspect of the invention, the peptide ofthe first aspect of the invention is coded by a nucleotide sequenceconsisting of a sequence having at least 90% of homology with one of thesequences of the group consisting of: SEQ ID NO. 7 to SEQ ID NO: 11. Inanother embodiment of the third aspect of the invention, the peptide ofthe first aspect of the invention is coded by a nucleotide sequenceconsisting a sequence having at least 90% of identity with one of thesequences of the group consisting of: SEQ ID NO. 7 to SEQ ID NO: 11. Inanother embodiment the peptide is coded by a nucleotide sequenceconsisting of a sequence selected from the group consisting of SEQ IDNO: 7 to SEQ ID NO: 11.

In another embodiment, the nucleotide sequence of the third aspect ofthe invention is operably linked to a promoter.

The term “operably linked to a promoter” as used herein refers to afunctional linkage between the promoter sequence and the sequence codingfor a fusion protein of the invention. This functional linkage isperformed through the fusion of a DNA sequence corresponding to anyMycoplasma pneumoniae promoter, or a promoter from other bacteriacompatible with M. pneumoniae, or a designed promoter capable of drivingexpression in M. pneumoniae, with the DNA sequence of the fusion protein(secretion peptide+POI) as is evident to any skilled person in the art.In principle the distance between the beginning of transcription afterthe promoter and the initial Methionine codon of the secretion signalshould be less than 100 bases and preferably less than 50 bases. Thepromoter sequence initiates and mediates transcription of the DNAcorresponding to the fusion protein coding sequence.

The selection of the promoter is made in view of the particular hostcell and also in view of different variables involved in the productionof the desired protein, such as inducible or constitutive expression,for instance. The skilled person in the art, using the generalknowledge, is able of selecting the more appropriate promoter.

In another embodiment, the nucleotide sequence of the third aspect ofthe invention is operably linked to a promoter, the promoter typicallybeing located upstream of the transcription start codon.

In still another embodiment, the nucleotide sequence of the third aspectof the invention is operably linked to an EfTu promoter. As it is shownbelow, EfTu promoter has been revealed by the experimental results togive high expression efficiency when the host cell is M. pneumoniae.

EfTu promoter sequence is available in several databases, The sequenceused in the present invention is SEQ ID NO: 12:

GAAGACCTTTTGTGCTAACGCCAGTTTGGCAAATCAAGTTCTGATTTTGCAATTATTTTGCTCCATATGAATTACACTACTCCAAGAATTATAAGCCTCTCTACAGCTTTATCTCAAACTTATGTAAAATTAGAGACGTAATTCAAACAC

In still another embodiment, the nucleotide sequence of the third aspectof the invention is operably linked to EfTu promoter, which is upstreamof the transcription start codon.

In a fourth aspect the present invention provides a vector comprisingthe nucleotide sequence of the third aspect of the invention coding forthe fusion protein.

The incorporation of the nucleotide sequence of the third aspect of theinvention in the vector is performed via any of the routine protocolsKnown to anyone skilled in the field of molecular biology.

In one embodiment of the fourth aspect of the invention, the vector isof the minitransposon Tn4001 type (Chopra-Dewasthali, R., et al. “Firststeps towards the genetic manipulation of Mycoplasma agalactiae andMycoplasma bovis using the transposon Tn4001mod” Int. J. Med. Microb.2005, vol. 294, pp. 447-453)

In one embodiment of the fourth aspect of the invention, the vector isof the mini-Tn4001PsPuro (GenBank accession no. FJ872396).

In another embodiment of the fourth aspect of the invention, the vectoris of minitransposon Tn4001PsPuro type and the nucleotide sequence isone comprising (a) a sequence having at least a 90% of homology with asequence of the group consisting of: SEQ ID NO 7 to SEQ ID NO: 11, and(b) a sequence coding for the POI. In another embodiment of the fourthaspect of the invention, the vector is of the same type as cited above,and the nucleotide sequence is one comprising (a) a sequence having atleast a 90% of identity with a sequence of the group consisting of: SEQID NO 7 to SEQ ID NO: 11, and (b) a sequence coding for the POI.

In another embodiment of the fourth aspect of the invention, the vectoris of the minitransposon Tn4001PsPuro type and the nucleotide sequenceis one comprising (a) a sequence having at least a 90% of homology witha sequence of the group consisting of: SEQ ID NO 7 to SEQ ID NO: 11, (b)a sequence coding for the POI(s); and (c) EfTu promoter located upstreamof the transcription start codon. In another embodiment of the fourthaspect of the invention, the vector is of the same type as cited aboveand the nucleotide sequence is one comprising (a) a sequence having atleast a 90% of identity with a sequence of the group consisting of: SEQID NO 7 to SEQ ID NO: 11, (b) a sequence coding for the POI(s); and (c)EfTu promoter located upstream of the transcription start codon.

In another embodiment of the fourth aspect of the invention, the vectoris of minitransposon Tn4001PsPuro type and the nucleotide sequence isone comprising (a) a sequence having at least a 90% of homology with asequence of the group consisting of: SEQ ID NO 7 to SEQ ID NO: 11, and(b) a sequence coding for the POI. In another embodiment of the fourthaspect of the invention, the vector is of the minitransposonTn4001PsPuro type and the nucleotide sequence is one comprising (a) asequence having at least a 90% of identity with a sequence of the groupconsisting of: SEQ ID NO 7 to SEQ ID NO: 11, and (b) a sequence codingfor the POI.

In another embodiment of the fourth aspect of the invention, the vectoris of minitransposon Tn4001PsPuro type and the nucleotide sequence isone comprising (a) a sequence having at least a 90% of homology with asequence of the group consisting of: SEQ ID NO 7 to SEQ ID NO: 11, (b) asequence coding for the POI(s); and (c) EfTu promoter located upstreamof the transcription start codon. In another embodiment of the fourthaspect of the invention, the vector is of the minitransposonTn4001PsPuro type and the nucleotide sequence is one comprising (a) asequence having at least a 90% of identity with a sequence of the groupconsisting of: SEQ ID NO 7 to SEQ ID NO: 11, (b) a sequence coding forthe POI(s); and (c) EfTu promoter located upstream of the transcriptionstart codon.

In other embodiment of the fourth aspect of the invention, the vectorcomprises other regulatory elements for improving the expression of thefusion protein, such as ribosomal binding sites, transcription start andtermination sequences, translation initiation sites, co-expression ofother polypeptides that can be used for later selection of clones(reporter genes). The vector can for instance be constructed in such away as to allow the controlled production of the fusion protein underspecific circumstances, such as for instance the presence or absence ofan inductor.

In another aspect, the present invention provides a host cell comprisingthe fusion protein of the second aspect of the invention or the vectorof the fourth aspect of the invention.

The term “host cell” as used herein is a prokaryotic cell that has beengenetically engineered so that it expresses the fusion protein of theinvention. In a particular embodiment, the “host cell” is a prokaryoticcell into which one or more vectors or isolated and purified nucleicacid sequences of the invention have been introduced. It is understoodthat it refers not only to the particular subject cell but also to theprogeny or potential progeny of such a cell. Because certainmodifications may occur in succeeding generations due to eithermutations or environmental influences, such progeny may not, in fact, beidentical to the parent cell, but are still included within the scope ofthe term as used herein.

The host cell of the invention can be used as a “factory” of the POI ofinterested, being cultured in vitro under adequate conditions (theconditions will depend on the specific cell). In this embodiment, thecell is grown in an appropriate medium under adequate conditions and itwill start the expression of the fusion protein, ie. the POI fused tothe peptide. Thanks to the signal peptide of the invention, the POI willbe efficiently exported through the membrane.

Alternatively, if selected host cell is intended to be administered to ahuman being or non-human animal as therapeutic tool in such a way thatPOI is produced in situ, and it is either unsafe or toxic, the selectedhost cell has to be engineered previous to its transformation with thevector of the present invention in order to develop a safe non-toxichost cell.

In a further aspect the present invention provides a pharmaceutical orveterinary composition.

The term “pharmaceutically or veterinary acceptable” refers toexcipients or carriers to be used in pharmaceutical or veterinarytechnology, for preparing compositions for medical use, either in humanbeings or animals. Each one of the components has to be acceptable fromthe pharmaceutical or veterinary point of view, having to be compatiblewith the other ingredients of the pharmaceutical or veterinarycomposition. It has to be also for use in contact with tissue or humanor animal organs without giving rise to excessive toxicity, irritation,allergic response, immunogenicity or other problems or side-effects. Theexpression “therapeutically effective amount” as used within the contextof the present invention, refers to the amount of a compound or a hostcell that, when administered, is sufficient to prevent development of,or alleviate to some extent, one or more of the symptoms of the diseasewhich is addressed. The particular dose administered according to thisinvention will of course be determined by the particular circumstancessurrounding the case, including the host cell administered, the fusionprotein that it produces, the route of administration, the particularcondition being treated, and similar considerations.

The fusion protein of the second aspect of the invention can forinstance be used for a variety of therapeutic applications. If the POIis a foreign protein antigen, the fusion protein can be used foreliciting an immune response against it, as long as the presence of thepeptide signal (secretion enhancer) does not alter its structure andfunction. Alternatively, if the POI is a therapeutic protein known tohave a beneficial effect, the fusion protein can be used in thetreatment of disease.

In a particular embodiment, the host cell is a bacterial host cell. Inanother embodiment, the host cell is an infectious bacteria. In anotherparticular embodiment of the fifth aspect of the invention, thebacterial host cell belongs to the genus Mycoplasma. In anotherparticular embodiment of the fifth aspect of the invention, thebacterial host cell is Mycoplasma pneumoniae. In another embodiment, itis a safe non-toxic host cell, either of wild-type or engineered. Inanother embodiment, it is a safe non-toxic engineered host cell. Inanother embodiment, it is a safe non-toxic engineered bacterial hostcell.

M. pneumoniae is an ideal starting point for designing a minimal cellfor use as bacterial therapy chassis. It has a small (860 kb) andcomprehensively annotated genome, and a rich collection of functionalgenomic data describing its transcriptome, methylome, proteome, andmetabolome. A flux balance model describing its metabolism is alsoavailable. In addition, it is closely related to M. genitalium and M.mycoides whose genomes have been chemically synthetized, transplanted,and comprehensively modelled. Furthermore, M. pneumoniae is well-suitedfor bacterial therapy because it is a weak human lung pathogen, dividesvery slowly (8-20 h), there are non-pathogenic strains, it does not havea lipolisacharide envelope (LPS) and is easily treated with commerciallyavailable antibiotics.

As it has been stated above, the fusion protein of the second aspectretains POI's function. When POI is a therapeutic polypeptide, thefusion protein or even the host cell of the fifth aspect of theinvention can be used in therapy.

The host cell of the fifth aspect of the invention can be formulated insuch a way as to improve its administration into different tissues.

In a particular embodiment of the seventh aspect of the invention, thefusion protein or the host cell is for use in the treatment of pulmonarydisease.

The former two embodiments can be reformulated into a method oftreatment of a disease comprising the administration of the fusionprotein of the second aspect of the invention or the host cell of thefifth aspect of the invention in a therapeutically effective amount to asubject in need thereof, and to a method of treatment of a pulmonarydisease comprising the administration of the fusion protein of thesecond aspect of the invention or the host cell of the fifth aspect ofthe invention in a therapeutically effective amount to a subject in needthereof, respectively.

Furthermore, the transformed bacteria could also be used for thedispersion of bacterial biofilms. As it is well-known, bacteria arecapable of sticking to surfaces with the help of a biofilm. That is,they produce and secrete a number of extracellular polymeric substances(basically proteins and polysaccharides) that form a matrix that allowsthem to colonize both organic and inorganic substrates. This matrix notonly serves as a fixation means, but also acts as a barrier ofteneffectively turning the bacteria into antibiotic resistant. It can bedevised that the host cells of the present invention could find boththerapeutic and non-therapeutic uses related to the destruction ofbacterial biofilms. The peptides of the present invention can be fusedto a series of hydrolysing enzymes capable of breaking down thepolysaccharide matrix. These fusion proteins can find both therapeuticand non-therapeutic applications. Therefore, it is also part of theinvention:

-   (i) the host cell of the fifth aspect for use in the degradation of    a bacterial biofilm. This applies for instance, to cystic fibrosis    or any other pulmonary disease that involves biofilm formation.-   (ii) the use of the host cell of the fifth aspect of the invention    for the degradation of bacterial biofilms (for example to clean    medical devices like cateters where biofilms could be found).

Throughout the description and claims the word “comprise” and variationsof the word, are not intended to exclude other technical features,additives, components, or steps. Furthermore, the word “comprise” andits variations encompasses the term “consisting of”. Additional objects,advantages and features of the invention will become apparent to thoseskilled in the art upon examination of the description or may be learnedby practice of the invention. The following examples are provided by wayof illustration, and they are not intended to be limiting of the presentinvention. Furthermore, the present invention covers all possiblecombinations of particular and preferred embodiments described herein.

EXAMPLES A) Material and Methods

To produce and secrete therapeutic proteins, inventors analysed thesecretome of M. pneumoniae. Then, they used this knowledge to build aseries of vectors to produce and secrete recombinant therapeuticproteins in M. pneumoniae. Normal M. pneumoniae media is rich inproteins which interfere with MS analysis. Therefore inventors used theminimal media of M. pneumoniae as basic growth media for our experiment(Yus, E. et al. “Impact dof genome reduction on bacterial metabolism andits regulation”, Science 2009, vol. 326, pp. 1263-1268) and replacedbovine serum albumin (BSA) as lipid carrier with(2-hydroxy)propyl-β-cyclodextrin (Hyprop).

After growing M. pneumoniae cells during 24 h and 72 h the supernatantmedia was harvested and ratios of intracellular to extracellular proteinconcentrations were obtained by dimethyl labelling and mass spectroscopyanalysis. On average inventors obtained ratios for 281 out of 688proteins from M. pneumoniae per experiment.

The proteins with highest extracellular to intracellular ratios werefurther analysed by the algorithm Signal P 3.0. Based on the predictedsecretion signals inventors designed 11 vectors comprising a promoterand a secretion signal fused to the therapeutic proteins. As negativecontrol inventors fused the 50 N-terminal residues of a cytosolicprotein (Mpn332) to the therapeutic proteins. As promoter inventorschose either the promoter corresponding to the coding sequence of thesecretion signal or if it was part of an operon with a distal promoterinventors put it under the control of the EfTu promoter. A detailed listof all constructs made, indicating the signal peptide and promoters canbe found In Tables 1-3 found below for Alginate lyase, p53 and A1AT.

TABLE 1 Secretion constructs and strains with alginate lyase A1-IIISignal Signal Signal Promoter length Name strain peptide Type PromoterLength [Bp] [AA] Cargo Mpn142(Opt)-A1-III Mpn142(Opt) SpI EfTu 150 31 AAA1-III Mpn142-A1-III Mpn142 SpI EfTu 150 31 AA A1-III Mpn152-A1-IIIMpn152 SpII Mpn152 106 33 AA A1-III Mpn200-A1-III Mpn200 SpII Mpn200 19728 AA A1-III Mpn213-A1-III Mpn213 SpI EfTu 150 34 AA A1-IIIMpn332-A1-III Mpn332 Neg EfTu 150 50 AA A1-III control Mpn400-A1-IIIMpn400 SpI Mpn400 160 41 AA A1-III Mpn489-A1-III Mpn489 SpII Mpn489 10034 AA A1-III Mpn506-A1-III Mpn506 SpII Mpn506 117 28 AA A1-IIIMpn588-A1-III Mpn588 SpI Mpn588 100 30 AA A1-III Mpn592-A1-III Mpn592SpII Mpn592 100 36 AA A1-III Mpn645-A1-III Mpn645 SpII EfTu 150 31 AAA1-III

TABLE 2 Secretion constructs and strain with A1AT Signal Signal SignalPromoter length Name strain peptide Type Promotor length [Bp] [AA] CargoMpn142(Opt)-A1AT Mpn142(Opt) SpI EfTu 150 Bp 31 AA A1AT Mpn142-A1ATMpn142 SpI EfTu 150 Bp 31 AA A1AT Mpn200-A1AT Mpn200 SpII Mpn200 197 Bp28 AA A1AT Mpn332-A1AT Mpn332 Neg EfTu 150 Bp 50 AA A1AT controlMpn400-A1AT Mpn400 SpI Mpn400 160 Bp 41 AA A1AT Mpn489-A1AT Mpn489 SpIIMpn489 100 Bp 34 AA A1AT Mpn506-A1AT Mpn506 SpII Mpn506 117 Bp 28 AAA1AT Mpn645-A1AT Mpn645 SpII EfTu 150 Bp 31 AA A1AT

TABLE 3 Secretion constructs and strain with p53 Signal Signal SignalPromoter length Name strain peptide Type Promoter Length [Bp] [AA] CargoMPN142-p53 Mpn142 SpI EfTu 150 31 AA p53 MPN200-p53 Mpn200 SpII Mpn200197 28 AA p53 Mpn332-p53 Mpn332 Neg EfTu 150 50 AA p53 controlMPN400-p53 Mpn400 SpI Mpn400 160 41 AA p53 MPN489-p53 Mpn489 SpII Mpn489100 34 AA p53 MPN506-p53 Mpn506 SpII Mpn506 117 28 AA p53 MPN459-p53Mpn459 SpI EfTu 150 57 AA p53 MPN645-p53 Mpn645 SpII EfTu 150 31 AA p53

The tables also indicate whether the secretion signals are predicted tobelong to membrane anchored lipoproteins (SpII) or sec mediated secretedproteins (SpI).

Inventors cloned the three therapeutic proteins fused to the secretionpeptide and the respective promoter in a miniTn4001-Puro-1 vector(GenBank accession number: KC816623). The constructs were assembled andscaled up in E. coli before transformation in M. pneumoniae. Inventorsdetermined the levels of the secreted protein either by an activityassay for the alginate lyase constructs or by ELISA measurements forA1AT and p53. In all cases, the constructs under the control of the EfTupromoter showed the highest level of secreted protein, while no signalwas detected for the negative control.

Inventors designed a battery of sequences that when fused toheterologous proteins result in their efficient expression andsecretion. To validate the vector sequences, inventors selected threedifferent proteins having possible therapeutic applications anddifferent biochemical and folding properties: an enzyme exemplified bypoly M Alginate lyase A1-III (alginase), an oligomeric protein: p53(which folds as a tetramer) and a protein with a difficultfold:□1-antitrypsin (A1AT).

Bacterial Strains and Growth Conditions

For constructing DNA vectors and secretion constructs different E. colistrains were used. Mainly Top10 (life technologies), Stbl4 (lifetechnologies) or copy cutter cells (epicentre). The cells were grown at30° C.-37° C. in LB or 2×TY medium with 100 ug/ml of Ampiciline forselection.

M. pneumoniae M129 strain was grown in T150 flasks at 37° C. in modifiedHayflick medium as previously described (Yus, E., et al. ibid). Whenselecting for Puromycin resistance 3 μg/ml were used. For the proteomicsstudies of the secreted proteins the cells were grown in modifiedminimal media. To make the minimal media compatible with MS analysis thelipid carrier BSA was exchanged to 5 mM cyclodextrin Hydroxypropyl ofestimated mol weight 1396Da (Sigma H107).

Experimental Determination of the Secretome

M. pneumoniae is usually cultured in modified Hayflick media, a richmedia containing many proteins from added horse serum. The highlyabundant proteins from the rich media cover the signal from low abundantsecreted proteins leaving them unsuitable for mass spectrometric (MS)analysis.

Therefore, inventors used the minimal media of M. pneumoniae as growthmedia for our experiment (Yus et al., ibid). This minimal media stillcontains bovine serum albumin (BSA) as lipid carrier in high amounts.Inventors replaced BSA with 5 mM (2-hydroxy)propyl-β-cyclodextrin(Hyprop) (Sigma H107 CAS Number 128446-35-5) (Greenberg-Ofrath et al.,“Cyclodextrins as carriers of cholesterol and fatty acids in cultivationof mycoplasmas” Appl. Environ. Microbiol. 1993, vol. 59, pp. 547-551)and could so obtain a protein free Media compatible with downstream MSanalysis.

To produce our supernatants inventors grew wt M129 in normal media for 3days. Before splitting the cultures were washed twice with PBS whileattached and twice after scraping. Inventors then split it 1:10 in a 150cm2 flask containing 40 ml of Hyprop media. Inventors started at eachrepeat of the experiment two flask one for each time point. The cellswere allowed to attach for 24 h and were the attached cells were washedtwice again with PBS to remove all trace amounts from the horse serum.Inventors then let the cells grow another 72 h before removing thesupernatant and harvesting the cells of the first flask. The attachedcells were resuspended in exactly the same amount of Hyprop media as theremoved supernatant. In the other flask only the supernatant was removedand the attached cells were washed twice with PBS before 35 ml of freshmedia was added. After 24 h this flask was harvested as the first one.

The cell suspension was always processed identical and in parallel tothe supernatants to avoid any bias from experimental procedure on theoutcome. The samples were precipitated with 60% acetone (Sigma product #179124) and 10% trichloracetic acid (TCA) (Sigma product # T9159) asfinal concentration. The mixture was spun for 1 h at 35000 g (4° C.).The supernatant was discarded and the pellets resuspended in 1.5 ml ofTCA/acetone and spun 2 h at 16000 g (4° C.). The supernatant was removedand the pellet was dried completely in a speed vac before beingredisolved in a buffer of 8 M Urea and 100 mM NaHCO3 using a bioruptorsystem. The total protein amounts in the samples were determined usingBCA assay (Pierce product # 23225). The UPF-CRG Proteomics facility thennormalised, digested and labelled the samples. Inventors used dimethyllabelling to label the different samples. Three Labels were used heavy,medium and light. Equal amounts of the samples mixed and analysed bynano LC/MS/MS to obtain ratios of intracellular to extracellular proteinconcentrations as previously described (Boersema et al., “Multiplexpeptide stable isotope dimethyl labelling for quantitative proteomics”Nat. Protoc. 2009, vol. 4, pp. 484-494). Inventors calculated thep-Value of a bimodal distribution by standard methods. Inventors chose aconservative p-value of 0.001 as threshold to define a protein assecreted.

Molecular Biology Methods

All constructs were cloned in mini-Tn4001-Puro vector (GenBank accessionnumber: KC816623). First, inventors generated a set of vectorscontaining the secretion signals and promoters. All fragments comprisingdifferent secretion signals and promoters were amplified by PCR from M.pneumoniae genomic DNA. For the secretion signals carrying their ownpromoter inventors introduced the PstI and EcoRI sites for restrictioncloning during the PCR and cloned the fragment by fast ligation. In thecases when the secretion signal had no own promoter and the EfTupromoter was used, inventors generated the constructs by the isothermalassembly method. Overhangs of 20 bp needed for the assembly wereintroduced by PCR. Detailed sequences of primers used are listed inTable 4, found below:

TABLE 4 Name Sequence Mpn152 RevGTGTGCCTGCAGGCTgCCATCAACTTGGTTAAATTTGCCCCTTGCC Mpn152 fwdAAGCTTGATATCGAATTCGCTTTTAAAAATACTTTTACTTCAGTAACTC AAAC Mpn200 Sig FAAGCTTGATATCGAATTCGACAGTAGTTTAAACTGATTCTTTACCTC Mpn200 Sig RGTGTGCCTGCAGGCTgCCTTTACCGCGTGTACCACAAG Mpn400 Sig FAAGCTTGATATCGAATTCGCGTAAATTTTCTCCTTTAGGGATACT Mpn400 Sig RGTGTGCCTGCAGGCTgCCATTAACGATAGAACTGCGGAAAAAGCA Mpn489 Sig FAAGCTTGATATCGAATTCTTCACCTTCACCTATTTTATTAGC Mpn489 Sig RGTGTGCCTGCAGGCTgCCATTGGAGGTATTGAGTGC Mpn506 Sig FAAGCTTGATATCGAATTCGATTAAATTTTCATCTTAAAAGCTTTTATTT TTACC Mpn506 Sig RGTGTGCCTGCAGGCTgCCTTTACCCTTTGTACCACAGGCAGC Mpn588 Sig FAAGCTTGATATCGAATTCTTCAATTAATCATTGATGGTTTAAGTGTCTC Mpn588 Sig RGTGTGCCTGCAGGCTgCCAAAGTTTGGCTGGGTTGCCAG Mpn592 Sig FAAGCTTGATATCGAATTCAACAGACCTTTAGAAGAAGTGCGA Mpn592 Sig RGTGTGCCTGCAGGCTgCCATTTTTGTGATTAGTGTTAGCTACTGTTAG CGT Mpn142 onlyTAGAGACGTAATTCAAACACATGAAATCGAAGCTAAAGTTAAAACGT signal_No1 TATTTACTGTTTTMpn142 only TGTTGGCTAGTGACAACGTCCCTAGCGGTAAAAGTGGTAAAAACAGT signal_No2AAATAACGTTTTA Mpn142 onlyGACGTTGTCACTAGCCAACACCTACCTCCTCCAAGGcAGCCTGCAGCC signal_No3 CGGGGGGCAAGAMpn645 only GTAGCTGCTGAAGAACAAGCGCTTAACAACAAACTAGAACCTAAAAG signal_No2AGAAATTAATAGAA Mpn645 onlyCGCTTGTTCTTCAGCAGCTACTCAAGTAATTTCTGGcAGCCTGCAGCC signal_No3 CGGGGGGCAAGAMpn645 only TAGAGACGTAATTCAAACACATGAAACTGAAACTTAAATTTCTATTAA signal_No1TTTCTCTTTTAG eFTu Fwd AAGCTTGATATCGAATTCGAAGACCTTTTGTGCTAACGCCAGeFTu Rev GTTTTGAATTACGTCTCTAATTTTACATAAGTTTG Mpn459 FGACGTAATTCAAACACATGGCTTTCATGCCATGTTTTTCATATAGC Mpn459 RevGTGTGCCTGCAGGCTgCCAGTAACATAAACATCTCGTGCTTGGGC Mpn213 RevGTGTGCCTGCAGGCTgCCTTGGTGGGCTTATTTACAAGCGTTATAGT TGTAGG Mpn213 FGACGTAATTCAAACACATGAAGCTTAGTGCTATTATCTCCCTATCAGT CG Mpn332ATGCCAGCTGTAAAAA Mpn332 GACTAACACCAAACGTTT

The resulting vectors derived from mini-Tn4001-Puro vector werelinearized using the restriction enzymes EcoRI (NEB R0101) and Pstl-Hf(NEB R3140) and subsequently dephosphorylated using Antartic phosphatse(NEB M0289). The different therapeutic proteins were synthetized andcloned into the EcoRI and Pstl-Hf digested vectors.

Mpn142 Opt (Design and Cloning)

The secretion signal of Mpn142 (First 93 coding bp) was ordered from DNA2.0, the company changed the codon choice to reduce secondary structurein the region of the secretion signal. Inventors then cloned thissequence as fusion to the target proteins. For the cloning inventorsused Gibson cloning and eliminated the previously used PstI restrictionsite.

Several publications showed that the secondary structure at the 5′ endof an mRNA influences the transcriptional efficiency. Recently, it hasbeen shown that this effect is emphasized on mRNA without a clearlydefined RBS site. Most transcripts of M. pneumoniae lack an RBS site andall constructs for production and secretion of alginate lyase weredesigned without RBS site. Inventors therefore hypothesized that areduction of mRNA secondary structure in the secretion signal couldimprove the translational efficiency.

Expression and Assessment of Activity in Different Proteins. NeutrophilElastase Activity Assay.

Inventors measured the activity of A1AT indirectly through theinhibition of neutrophil elastase. The activity of neutrophil elastasewas measured either by a colorimetric or fluorescence based assay. Forthis the commercially available substrateN-Methoxysuccinyl-Ala-Ala-Pro-Val p-nitroanilide (Sigma product # M4765)or the cleavage of a quenched fluorescence substrateN-Methoxysuccinyl-Ala-Ala-Pro-Val-AMC (Merck millipore product # 324740)was used. The reaction buffer is 0.1 M Tris ph 7.5. In the colorimetricassay the release of free 4-nitroaniline was followed at 400 nm and inthe fluorescence assay an excitation of 370 nm was used and the emissionwas recorded at 445 nm.

Enrichment of A1AT for Neutrophil Elastase Activity Measurements

To measure concentrated minimal media supernatant, inventors firstimproved the culture condition to reduce the carryover of horse A1AToriginating in the pre culture to a minimum. Inventors grew apre-culture in full Hayflick medium, the media was aspirated and cellswere washed twice with PBS. Then, the cells were scraped and split 1:10in minimal media. The minimal media was aspirated and replaced withfresh media after 24 h. The culture was grown for 48 and then thesupernatant was harvested. To measure A1AT activity on concentratedmedia 200 μl of supernatant were reduced to a volume of 20 μl using anAmicon ultrafiltration device with a 30 kDa cut-off and then the A1ATactivity was tested.

To enrich A1AT by affinity chromatography inventors used the Strep-TagIIfused to A1AT. Inventors harvested supernatant from 300 cm2 culture dishwith cells grown in full Hayflick media. The media was run over a 1 mlStrepTrap HP column (GE Healthcare product # 28-9075-46). The column wasthen washed with 6 column volumes of wash buffer (100 mM Tris, pH 8.0,150 mM NaCl, 1 mM EDTA) and subsequently eluted with wash buffersupplemented with 2.5 mM desthiobiotin. The concentration of A1AT wasdetermined by ELISA.

P53 and A1AT Quantification by ELISA

Inventors used commercially available Kits for the quantification ofhuman A1AT (USCN Product No.: SEB697Hu) and a p53 pan ELISA (RocheProduct No.: 11828789001). For the assay inventors followed themanufacturer's instructions.

Alginate Lyase Assay

To measure alginate lyase activity in full media the assay developed byKitamikado, M., et al. “Method designed to detect alginate-degradingbacteria” Appl. Environ. Microbiol. 1990, vol, 56, pp. 2939-2940) wasused. Briefly, 0.1% of alginate substrate is added to the media and withthe cells. At various time points 0.2 ml of media supernatant is put ina test tube and 2.0 ml of an acidic albumin solution (3.26 g sodiumacetate, 4.56 ml of glacial acetic acid, 1.0 g of bovine albuminfraction V are filled up to 11 with water and ph adjusted to 3.75 withHCl). In the presence of polymeric alginate a white precipitate isformed. A small aliquot of the mixture is then transferred to a plateand the absorbance is measured.

Inventors tested different wavelengths for the signal to noise ratio andfound 300 nm to be the most sensitive, while everything up to 660 nmgave good reliable readings.

B) Results

Overall, the results presented here are the proof of concept that M.pneumoniae can be used as a delivery system to express and secreteactive proteins with a range of applications, including those related totherapy. They show that it is possible to engineer M. pneumoniae toproduce and secrete therapeutic proteins. Inventors could produce anddetect in the supernatant 3 different proteins, two of which are ofhuman origin (p53/A1AT) and of which only two are normally secreted(alginate lyase/A1AT). Inventors could prove that our secretion vectorworked reliably with the same construct yielding the highest yields forall three proteins. Further, we could show that two out of the threeproteins are correctly folded and active after secretion.

Quantitative Analysis of the Secreted Proteins by M. pneumoniae.

Inventors designed 11 vectors comprising a promoter and a secretionsignal fused to the three therapeutic proteins (A1AT, p53, Alginase). Asa negative control inventors fused a fragment of the same lengthcorresponding to the N-terminus of a cytosolic protein (Mpn332) to thethree proteins.

Quantitative analysis of the secreted p53 by M. pneumoniae Aftertransforming M. pneumoniae cells with each of the engineered p53constructs, p53 secretion was verified by using a p53 pan sandwich ELISA(Roche). To assess the dynamics of p53 accumulation in the supernatant,the absolute concentrations of p53 in the media was measured at varioustime points after inoculation. Inventors characterized protein secretionboth in modified Hayflick (Full Media) and McCoy media which is commonlyused for mammalian cell culture. The results for secretion in McCoymedia are summarized in FIG. 1. The constructs Mpn-142 and Mpn-645 undercontrol of the Eftu promoter showed the highest concentration of p53 inthe supernatant media. Interestingly the p53 concentration in thesupernatant for most constructs peaked at 48 h when the cells enter intostationary phase and then slowly dropped again. This could eitherindicate either a regulation of secretion or gene expression which islinked to the switch from exponential to stationary phases of growth.Also, a balance between secretion and extracellular breakdown byproteases could explain this result.

Quantitative and Functional Analysis of the Secreted Alginate LyaseAl-III by M. pneumoniae

Inventors further analysed the secretion and activity of alginate lyasein M. pneumoniae. First the gene was cloned in our secretion constructsand then transformed into M. pneumoniae. To evaluate the secretionefficiency inventors used an assay for alginate lyase activity. As withthe p53 constructs the two constructs with the alginate lyase undercontrol of the EfTu promoter and with the secretion signals of Mpn-142and Mpn-645 performed the best (FIG. 2). Both showed degradation of thealginate in the media already at day 1. While the majority of theconstructs showed no sign of alginate degradation even after 5 days ofincubation. The constructs with the secretion signals of Mpn-200 andMpn-400 which are both under their respective promoter showeddegradation of alginate at day 2 and the construct with the secretionsignal of Mpn-489 and its respective promoter showed degradation only atday 5 after inoculation. The negative control which is fused to the 50N-terminal amino acids of the cytosolic protein Mpn-332 showed no signof degradation. No differences in growth rate of different M. pneumoniaestrains were observed in comparison with wt indicating that the promoteractivity is a determining factor for the production and secretion ofalginate lyase.

In order to test if this system could be implemented in a lessimmunogenic strain, we tested whether the secretion constructs also workin a non-adherent strain. This strain lacks part of the adhesion andgliding machinery, main factors in virulence and pathogenicity. Theresults obtained in the non-adherent strain matched up with the resultsobtained in wt M129 strain implying that the same secretion vectors canbe used in non-pathogenic strain.

In order to improve the levels of protein production, inventors designeda new Mpn-142 secretion signal (Mpn-142(Opt) (which corresponds to SEQID

NO:7) with a minimized secondary structure in the 5′ of the mRNA. Theconstruct was still under the control of the EfTu promoter but thecodons for the secretion signal were changed from the wt sequencefollowing the recommendations of the company DNA 2.0. Inventors hadobserved earlier that M. pneumoniae grows better in Hayflick (full)media than in our modified minimal media used to characterize thesecretome. To quantify the maximal amounts of alginate lyase produced bythe different strains, inventors thus established a quantitative assaycompatible with full media based on the previously used qualitativeassay (Kitamikado et al., ibid). The absolute alginate lyase activityfrom the media of 2 strains was determined at different time points. Thehighest activity levels were observed for the Mpn-142(Opt) strain. Inthe media of the M. pneumoniae strain transformed with Mpn-142(Opt)inventors measured an activity corresponding to ˜0.1 Units of alginatelyase. This corresponds to ˜0.01 mg/ml of the alginate lyase (SigmaA1603) which we used as standard in our quantitative assay.

Inventors finally tested supernatants of the strains Mpn-142(Opt) andMpn-142 in a P. aeruginosa biofilm assay. Inventors saw no anti biofilmactivity while a positive control of media containing the same amount ofalginate lyase activity, of a commercial alginate lyase (Sigma A1603)showed antibiofilm activity.

It has been reported that DNAse enhances the breakdown of P. aeruginosabiofilms. Based on this study inventors investigated whether M.pneumoniae M129 media supernatant contains Dnase activity that couldalready supply this function. For this propose, inventors measuredlambda phage DNA degradation in the media from a M129 WT culture. After1 h of incubation at 37° C. most of the DNA was digested indicating astrong Dnase activity in the supernatant of M. pneumoniae M129 wtsupernatants. Only minor signs of degradation were observed in thecontrol reaction corresponding to the media that had not been in contactwith cells.

Quantitative analysis of the secreted A1AT by M. pneumoniae In order totest the activity of AA1T secreted by M. pneumoniae, first inventorsmeasured the protein expression level in the supernatant for each strainusing an ELISA kit from USCN. As shown in FIG. 3A a, secretion of AAT isbetter using full medium. The best secretion levels are obtained, as inthe previous cases, from the Mpn-142 and Mpn-645 strains with a yieldrespectively of 250 ng/ml and 130 ng/ml using the full medium and 152ng/ml and 20 ng/ml using the minimal medium after 73 h of culture. Aswith p53 inventors observed that the protein concentration in thesupernatant peaked after 2-3 days and then decreased (FIG. 3a ).Inventors tested whether the degradation of the secreted protein by anextracellular protease is the mechanism causing this peak. Therefore,inventors spiked supernatant of a wt M. pneumoniae culture grown eitherin minimal or full Hayflick media with 300 nM A1AT and incubated it for96 h. Inventors measured the inhibitory effect on neutrophil elastase atdifferent time points. All sample showed a constant maximal inhibitionindicating no significant degradation in the supernatant over time.Inventors performed a similar test with BSA in supernatant and couldalso observe no proteolytic effect on a SDS Gel.

Then inventors analyzed if the secreted A1AT was functional.Unfortunately the full Hayflick media which yields the highest proteinsconcentrations also contains high level of Horse A1AT. This Horse A1ATinterferes with functional neutrophil elastase assays while not showingup in the ELISA measurements The amounts of A1AT secreted in the minimalmedia is below the minimum concentration necessary for the functionalassay 10 to 30 nM (500 ng/ml-1500 ng/ml) (FIG. 3A). To reach theconcentration of 10 to 30 nM of AAT in the supernatant of Mpn-142culture, inventors concentrated the samples from minimal media usingultrafiltration with an ultra-30K device (Amicon). As shown in FIG.3bconcentrated supernatant showed a complete inhibition of NE activitycompared to WT concentrated supernatant. However, inventors observed adecrease of NE activity in the minimal media alone in comparison withour control reaction in buffer only suggesting unspecific inhibition ofNE activity following the volume reduction.

To further verify the activity of AAT secreted by M. pneumoniae, apurification process was performed. Inventors used a Strep Tag II whichwas introduced at C-terminus of the protein in conjunction with a StrepTactin column to enrich the protein. The binding of the protein to thecolumn was weak most likely because of the low concentration of A1AT inthe media compared to the Kd of the Strep Tag II. Neither the lessinventors could significantly enrich and purify the A1AT from the fullmedia. Inventors then tested the purified protein in a Neutrophilelastase activity test and could show that the A1AT produced by M.pneumoniae is as active as A1AT produced by E. coli (FIG.3c).

After transforming M. pneumoniae cells with each of the engineered p53,A1AT and alginase constructs, inventors verified secretion in thesupernatant in modified Hayflick (full media) at different time pointsusing different analyses. In the case of p53 secretion was verified byusing a p53 pan sandwich ELISA (Roche). In the case of the alginase,inventors checked the enzymatic activity (alginate degradation) in thesupernatant. Finally, for A1AT inventors checked the expression inminimal media as well due to the abundance of horse A1At in the medium.

In the three cases, similar results were found (FIG. 1-FIG. 3) with theconstructs Mpn-142 and Mpn-645 under the control of the EfTu promotershowing the highest concentration in the supernatant media. Nodifferences in growth rate of different M. pneumoniae strains wereobserved in comparison with WT indicating that the promoter activity isa determining factor for the production and secretion of the proteins.These results indicate that the selected P. pneumoniae secretion signalsworked similarly with very different proteins, and that: enzymes,oligomeric proteins or difficult folds can be effectively produced andreleased to the medium. For most constructs and cases the concentrationin the supernatant in rich medium peaked at 48h and then slowly droppedagain. This could either indicate a regulation of secretion or geneexpression which is linked to the switch from exponential to stationaryphases of growth. Also, a balance between secretion and extracellularbreakdown by proteases could explain this result.

Testing other strains and improving production.

In order to test if the secretion system could be implemented in anon-pathogenic strain, inventors tested whether the algynase constructscould also work in a non-adherent strain. This strain lacks part of theadhesion and gliding machinery, which are main factors in virulence andpathogenicity. The results obtained in the non-adherent strain matchedup with the results obtained in M129 strain implying that the samesecretion vectors can be used in non-pathogenic strains.

In order to improve the levels of protein production, inventors designeda new Mpn-142 secretion signal (Mpn-142(Opt)) with a minimized secondarystructure in the 5′ of the mRNA. The construct was still under thecontrol of the EfTu promoter but the codons for the secretion signalwere changed from the wt sequence. To quantify the maximal amounts ofalginate lyase produced by the different strains, a quantitative assaycompatible with full media based on a previously used qualitative assaywas established. The absolute alginate lyase activity from the media ofthe MPN142 and MPN142(Opt) strains was determined at different timepoints. The highest activity levels were observed for the Mpn-142(Opt)strain in the media of the M. pneumoniae strain transformed withMpn-142(Opt) inventors measured an activity corresponding to ˜0.1 Unitsof alginate lyase. This corresponds to ˜0.01 mg/ml of the alginate lyase(Sigma A1603) which was used as standard in the quantitative assay.

REFERENCES CITED IN THE APPLICATION

-   Chopra-Dewasthali, R., et al. “First steps towards the genetic    manipulation of Mycoplasma agalactiae and Mycoplasma bovis using the    transposon Tn4001mod” Int. J. Med. Microb. 2005, vol. 294, pp.    447-453-   Yus, E. et al. “Impact dof genome reduction on bacterial metabolism    and its regulation”, Science 2009, vol. 326, pp. 1263-1268-   Greenberg-Ofrath et al., “Cyclodextrins as carriers of cholesterol    and fatty acids in cultivation of mycoplasmas” Appl. Environ.    Microbiol. 1993, vol. 59, pp. 547-551-   Boersema et al., “Multiplex peptide stable isotope dimethyl    labelling for quantitative proteomics” Nat. Protoc. 2009, vol. 4,    pp. 484-494-   Kitamikado, M., et al. “Method designed to detect alginate-degrading    bacteria” Appl. Environ. Microbiol. 1990, vol, 56, pp. 2939-2940

1. A peptide comprising a sequence having at least 90% of homology withone of the sequences of the group consisting of: SEQ ID NO: 1; SEQ IDNO: 2; SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO:
 6. 2. Thepeptide of claim 1 comprising the sequence SEQ ID NO: 1, SEQ ID NO: 2;SEQ ID NO:
 3. 3. A fusion protein comprising a polypeptide of interestand at least one peptide as defined in claim 1, wherein the polypeptideof interest is heterologous to the at least one peptide.
 4. (canceled)5. The fusion protein of claim 3, wherein the polypeptide of interest isselected from the group consisting of p53, Alginate Lyase A1-III andα1-antitrypsin.
 6. A nucleotide sequence coding for the peptide ofclaim
 1. 7. The nucleotide sequence of claim 6 operably linked to apromoter.
 8. A vector comprising the nucleotide sequence of claim
 6. 9.A host cell comprising the vector of claim
 8. 10. The host cell of claim9, wherein the cell is a bacterial cell.
 11. (canceled)
 12. Apharmaceutical or veterinary composition comprising a therapeuticallyeffective amount of the fusion protein as defined in claim 3 which is asafe and non-toxic cell, together with pharmaceutically acceptableexcipients or carriers.
 13. A method of secreting a polypeptide ofinterest, said method comprising using the peptide of claim 1 as asecretion peptide of a polypeptide of interest.
 14. (canceled)
 15. Amethod of treating a pulmonary disease in a subject in need thereof,said method comprising administering the fusion protein of claim 3 in atherapeutically effective amount to the subject in need thereof.
 16. Anucleotide sequence coding for the fusion protein of claim
 3. 17. Thenucleotide sequence of claim 16 operably linked to a promoter.
 18. Avector comprising the nucleotide sequence of claim
 16. 19. A host cellcomprising the vector of claim
 18. 20. The host cell of claim 19,wherein the cell is a bacterial cell.
 21. The host cell of claim 20,wherein the bacterial host cell is Mycoplasma pneumoniae.
 22. Apharmaceutical or veterinary composition comprising a therapeuticallyeffective amount of the host cell as defined in claim 19, which is asafe and non-toxic cell, together with pharmaceutically acceptableexcipients or carriers.
 23. A method of treating a pulmonary disease ina subject in need thereof, said method comprising administering the hostcell of claim 19 in a therapeutically effective amount to the subject inneed thereof